Earlier we showed altered expression of Hsp40s, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain proteins affect prion propagation in wild type and Hsp70 mutant cells. We also identified several Hsp40 and TPR protein mutant alleles that impair or enhance prion propagation. Our data suggest that many, if not all, of the observed effects of co-chaperones on prions are mediated by their regulation of Hsp70 activities. In many instances the same conditions producing a significant effect on one prion have either little or an opposite effect on a different prion. These data point to a prion preference or specificity of the Hsp70/co-chaperone pairings. Targeting the two major yeast Hsp40s (Ydj1p and Sis1p), we earlier showed that while overexpressing Sis1p has no effect on prions, elimination of the URE3 prion by overproduced Ydj1p requires only its ability to regulate Hsp70. The curing efficiency also depended on particular isoforms of Hsp70. Therefore, prion curing was indirect and required interaction of specific Hsp40 with Hsp70, but not amyloid. Our other data show that while Hsp40s other than Sis1p can contribute to the prion replication mechanism, curing of prions by overexpressed Hsp104 is strictly dependent on Sis1p. These findings are consistent with prion propagation and elimination requiring different chaperone activities, and suggest specificity of Hsp104 functions can be controlled by the way Hsp70 interacts with its co-chaperones. We adapted our yeast system for studying prokaryotic chaperones and co-chaperones to determine whether various functions of chaperone machinery components are specific or overlapping. We found ClpB supports both prion propagation and thermotolerance in yeast if it is modified to interact with yeast Hsp70 or if E. coli Hsp70 and its cognate nucleotide exchange factor (NEF) are present. Different yeast Hsp40s play critical roles in these distinct processes. Our findings show prion propagation and thermotolerance minimally require Hsp100, Hsp70, NEF and Hsp40, with Hsp40 directing functions of this machinery. They also define cooperative interactions among these components that are specific or interchangeable across life kingdoms, and imply Hsp100 family disaggregases possess intrinsic amyloid remodeling activity.